CN106906213B - Modified siRNA and application thereof - Google Patents

Abstract

The invention discloses a modified siRNA and application thereof. According to the invention, gemcitabine is taken as an example of a siRNA modification monomer, and RRM1mRNA targeted siRNA is specifically and effectively modified to obtain a novel small molecular targeted compound Gem-RRM1siRNA, so that the proliferation of pancreatic cancer cells can be effectively inhibited, the effect of RRM1siRNA on reversing gemcitabine drug-resistant pancreatic cancer cells can be enhanced, and the effect of synergistically inhibiting tumors is achieved. It is foreseen that: the nucleoside analogue with biological activity or cytotoxicity can also be used as siRNA modified monomer, and after specific effective modification is carried out on specific siRNA, the nucleoside analogue and the siRNA have synergistic effect, and can be applied to development and preparation of disease treatment drugs.

Description

Modified siRNA and application thereof

Technical Field

The invention relates to the field of siRNA, in particular to modified siRNA and application thereof.

Background

siRNA is a double-stranded RNA 20 to 25 nucleotides in length that causes RNA interference by recognizing and binding to mRNA complementary to its sequence, causing degradation of the target mRNA, ultimately silencing the gene of interest. Since siRNA can directly interfere the function of key target at gene level, siRNA which does not need to rely on the crystal structure confirmation of target protein becomes a candidate molecule of small molecule targeted drug, and has shown huge potential and application prospect in the treatment of various diseases. Taking tumor treatment as an example, the siRNA drug can stably deliver siRNA to directionally enter tumor tissues, and accurately interfere cell signal pathways, thereby achieving the purpose of treating tumors.

However, there are some problems in the application of siRNA drugs, such as: siRNA is easily degraded by nuclease, has a very short half-life in vivo, cannot ensure that siRNA molecules reach the correct part of the body, and is liable to cause severe immune response in the body. In recent years, researches show that the siRNA is chemically modified, so that the stability of the siRNA in vivo can be greatly improved, the half-life period of the siRNA in cells and blood can be prolonged from several minutes to several days, and the immune response of the body to the siRNA and the off-target effect of the siRNA are reduced. For example, the drug ALN-AT3 obtained by Alynam's siRNA modified by galactosamine conjugation can stably and effectively deliver specific siRNA, which is approved by the U.S. FDA to treat hemophilia A in 8 months of 2013.

Currently, chemical modifications of siRNA mainly include modifications of the sugar group, backbone and end. The glycosyl is modified by common 2-hydroxyl modification modes such as methylation, methoxyethylation, fluoridation and the like, so that the stability of the siRNA can be improved, and the binding capacity of the siRNA and complementary mRNA can be enhanced; the main chain modification is mainly P-S modification, which can enhance the nuclease resistance while keeping the siRNA binding capacity, but researches show that excessive modification can cause serious toxic reaction; the modification of the siRNA terminal comprises polyethylene glycol modification, cholesterol modification and the like: the polyethylene glycol has the unique biological properties of reducing cytotoxicity, enhancing water solubility, shielding negative charges and the like; the cholesterol modification utilizes the characteristic that cholesterol can be combined with low-density lipoprotein, high-density lipoprotein and other in-vivo transfer carriers, improves the half life of siRNA, simultaneously promotes the absorption of siRNA by hepatocytes by combining with a low-density lipoprotein receptor expressed by the liver, and can be applied to the treatment of liver diseases.

Some nucleoside analogs have antitumor effects, such as first-line drug 5-FU for treating colon cancer, 5-azacytidine for treating hematological disorders, Gemcitabine (Gemcitabine, Gem for short) for treating pancreatic cancer, etc. Gemcitabine is a nucleoside antitumor drug commonly used in clinic at present, is widely applied to clinical treatment of various tumors and hematopathy, and is the only effective drug for treating pancreatic cancer at present. Unfortunately, patients develop resistance within weeks of gemcitabine use, and thus gemcitabine has very limited effectiveness in treating pancreatic cancer. Overexpression of the ribonucleotide reductase M1 (ribosomal reductase M1 subbunit, RRM1) is one of the major mechanisms for gemcitabine to develop drug resistance. Clinical and basic studies showed that levels of RRM1 correlate with patient survival, with higher levels of RRM1 resulting in lower patient survival; the study showed that RRM1 expression was inhibited and the sensitivity of the cells to gemcitabine increased.

Disclosure of Invention

The invention aims to provide a modified siRNA, which is obtained by modifying siRNA with a nucleoside analogue with bioactivity or cytotoxicity, wherein the nucleoside analogue with bioactivity or cytotoxicity is preferably gemcitabine or a gemcitabine structural analogue.

The invention also aims to provide application of the modified siRNA in preparing a tumor treatment medicament, in particular application of gemcitabine modified RRM1siRNA in preparing a tumor treatment medicament.

The technical scheme adopted by the invention is as follows:

a modified siRNA, said modification being:

the end of the initial siRNA is coupled with at least one nucleoside analogue monomer with biological activity or cytotoxicity;

inserting at least one nucleoside analogue monomer with biological activity or cytotoxicity into the sequence of the initial siRNA;

and at least one nucleotide within the sequence of the initial siRNA is replaced with a biologically active or cytotoxic nucleoside analog monomer.

Further to the above scheme, the initial siRNA is further coupled with at least one nucleotide at its end.

Preferably, the end of the sense strand or the antisense strand of the initial siRNA is coupled with 1-5 nucleoside analogue monomers with biological activity or cytotoxicity.

Preferably, the end of the sense strand or the antisense strand of the initial siRNA is also coupled with 3-6 nucleotides.

Preferably, the end of the sense strand or the antisense strand of the initial siRNA is coupled with 1-2 nucleoside analogue monomers with biological activity or cytotoxicity and 3-4 nucleotides.

Preferably, 1-3 nucleoside analogue monomers with biological activity or cytotoxicity are inserted into the sequence of the initial siRNA.

Preferably, 1-3 nucleotides within the sequence of the initial siRNA are replaced by a biologically active or cytotoxic nucleoside analog monomer.

Preferably, the biologically active or cytotoxic nucleoside analog is gemcitabine or a structural analog of gemcitabine.

Preferably, the initial siRNA is an siRNA targeting RRM1mRNA, the double-stranded nucleotide sequence of which is as follows:

sense strand: 5'-GGAAGAAGAUGAUAAAGAA-3' the flow of the air in the air conditioner,

antisense strand: 5'-UUCUUUAUCAUCUUCUUCC-3' are provided.

The modified siRNA is applied to the preparation of tumor treatment medicines.

The invention has the beneficial effects that:

the invention takes gemcitabine to modify siRNA of targeting RRM1mRNA as an example, obtains a novel small molecular targeting compound Gem-RRM1siRNA by specifically and effectively modifying, can not only effectively inhibit pancreatic cancer cell proliferation, but also enhance the effect of RRM1siRNA in reversing gemcitabine drug-resistant pancreatic cancer cells, and has the effect of synergistically inhibiting tumors.

It is foreseen that: the nucleoside analogue with biological activity or cytotoxicity can also be used as siRNA modified monomer, and after specific effective modification is carried out on specific siRNA, the nucleoside analogue and the siRNA have synergistic effect, and can be applied to preparation and development of disease treatment drugs.

Drawings

FIG. 1: detecting the specificity of 4 siRNAs by a western blot and qPCR method, (A) detecting the inhibition effect of 0nM and 10nM siRNAs on the intracellular RRM1 protein level; (B) the inhibition of cellular RRM1mRNA levels by 10nM siRNA was examined.

Detailed Description

The following examples are only for the purpose of helping the skilled person to better understand the present invention, but do not limit the present invention in any way.

Example 1 Gemcitabine-modified RRM1siRNA

First, design and optimization of siRNA

Based on different action sites of RRM1mRNA sequence (Gene ID:6240), through repeated tests and screening, a siRNA (hereinafter referred to as RM2) targeting RRM1mRNA with good specificity is selected, and the double-stranded nucleotide sequence is as follows:

sense strand: 5'-GGAAGAAGAUGAUAAAGAA-3' (SEQ ID NO: 1)

Antisense strand: 5'-UUCUUUAUCAUCUUCUUCC-3' (SEQ ID NO: 2)

RM2 was subjected to conventional chemical modification, i.e., RM2 with 2 dT coupled to the 3 'end of the sense strand and dAdT coupled to the 3' end of the antisense strand to obtain conventionally modified RM2, 10nM was transfected with Lipofectamin 2000(Invitrogen), and after transfection into cells at a 3: 1 ratio of Lipofectamin 2000 to siRNA mass, Western blot was used to examine the effect of siRNA on RRM1 protein expression, and qRT-PCR was used to examine the effect of siRNA on RRM1mRNA level, and negative control (misatchRNA) and positive control (control) were set.

The results are shown in fig. 1A, conventional modified RM2 has a significant inhibitory effect on intracellular RRM1 protein at 10nM dose; as shown in figure 1B, RM2 decreased RRM1mRNA expression by 85% compared to control. Description of the drawings: RM2 has high specificity, and can be well combined with mRNA of RRM1 to degrade the mRNA, thereby inhibiting the expression of protein.

Second, gemcitabine modified RRM1siRNA

RM2 was used as the initial siRNA to be modified, and gemcitabine was used for the following modification to obtain Gem-RRM1 siRNA.

The modification strategy is as follows: coupling 3-4 nucleotides at the 3' end of the sense strand or antisense strand of the initial RRM1siRNA, and coupling 1-2 gemcitabine monomers (such as RRM-RRM 1siRNA with the number of 1-6); coupling 5 gemcitabine monomers (such as 7-8 Gem-RRM1siRNA) at the 3' end of the sense strand or antisense strand of the initial RRM1 siRNA; 1-3 gemcitabine monomers (e.g., Gem-RRM1siRNA, accession No. 9-11) were inserted or/and substituted within the sequence of the original RRM1siRNA with nucleotide-modified siRNA alone as a control (e.g., accession No. 12) and gemcitabine-modified mispatch siRNA as a control (e.g., accession No. 13).

The more monomers used for siRNA modification, the better the modification, and each modified product is specific, and a luciferase experiment is needed to detect the biological effectiveness of the modified siRNA. Modified RRM1siRNA sequences were provided according to the protocol described above, as shown in table 1.

TABLE 1 Gemcitabine modified siRNA sequences

Note: in the table, X represents gemcitabine, and the right side of "-" is a specific sequence for modifying the monomer composition.

Third, WST-1 experiment evaluation Gem-RRM1siRNA inhibits drug-resistant pancreatic cancer cell PANC1/Gem proliferation

After gemcitabine drug-resistant pancreatic cancer cells PANC1/Gem are inoculated into a 96-well plate for overnight culture, the 13 modified siRNAs are transfected by Lipofectamin 2000(Invitrogen), the modified siRNAs are transfected into cells according to the mass ratio of Lipofectamin 2000 to siRNA being 3: 1, and IC is detected by a WST-1 cell proliferation and cytotoxicity detection kit after 72 hours₅₀。

TABLE 2 Effect of Gem-RRM1siRNA on proliferation of drug-resistant pancreatic cancer cells PANC1/Gem

The results are shown in Table 2, compared with gemcitabine-modified mismatch siRNA control (No. 13), the inhibition effect of Gem-RRM1siRNA with the numbers of 1-11 on drug-resistant pancreatic cancer cells PANC1/Gem is very obvious, and RRM1siRNA can reverse the drug-resistant effect of gemcitabine. Compared with the siRNA control (number 12) modified by a conventional siRNA modified monomer, the Gem-RRM1siRNA with the numbers of 1-7 and 10-11 has more obvious inhibition effect on drug-resistant tumor cells PANC1/Gem, which shows that modification of siRNA by gemcitabine can not only effectively inhibit proliferation of pancreatic cancer cells, but also enhance the effect of RRM1siRNA on reversing drug resistance, and has the effect of synergistically inhibiting tumors. Among the modified siRNAs, RM2-2 XG (2.3) -dU/dA has the strongest effect, and RM2-2GdU/dA has the second time, and the modified siRNAs which can effectively reverse drug resistance and synergistically inhibit cancer are expected to be used for developing tumor gene therapeutic drugs.

Thus, we can foresee: nucleoside analogs with biological activity or cytotoxicity can also be used as siRNA modifiers, and can be applied to the development and preparation of disease treatment drugs after specific effective modification of specific siRNA.

SEQUENCE LISTING

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Claims (2)

1. A modified siRNA, comprising: the modification is as follows:

the end of the initial siRNA is coupled with at least one nucleoside analogue monomer with biological activity or cytotoxicity;

inserting at least one nucleoside analogue monomer with biological activity or cytotoxicity into the sequence of the initial siRNA;

and at least one nucleotide within the sequence of the initial siRNA is replaced with a biologically active or cytotoxic nucleoside analog monomer;

1-2 nucleoside analogue monomers with biological activity or cytotoxicity and 3-4 nucleotides are coupled at the tail end of the sense strand or the antisense strand of the initial siRNA;

inserting 1-3 nucleoside analogue monomers with biological activity or cytotoxicity into the sequence of the initial siRNA;

1-3 nucleotides in the sequence of the initial siRNA are replaced by nucleoside analogue monomers with biological activity or cytotoxicity;

the nucleoside analogue monomer with bioactivity or cytotoxicity is gemcitabine;

the initial siRNA is an siRNA targeting RRM1mRNA with the following double-stranded nucleotide sequence:

sense strand: 5'-GGAAGAAGAUGAUAAAGAA-3' the flow of the air in the air conditioner,

antisense strand: 5'-UUCUUUAUCAUCUUCUUCC-3' are provided.

2. Use of the modified siRNA of claim 1 for the preparation of a medicament for the treatment of pancreatic cancer.

Images